Actively targeted low-dose camptothecin as a safe, long-acting, disease-modifying nanomedicine for rheumatoid arthritis

Abstract

Purpose: Camptothecin (CPT), a potent topoisomerase I inhibitor, was originally discovered as an anticancer agent to induce programmed cell death of cancer cells. Recent evidence suggests that, similar to cancer, alterations in apoptosis and over-proliferation of key effector cells in the arthritic joint result in rheumatoid arthritis (RA) pathogenesis. Initial in vitro studies have suggested that camptothecin inhibits synoviocyte proliferation, matrix metalloproteinases expression in chrondrocytes and angiogenesis. This study is one of the first to test, in vivo, RA as a new indication for CPT.

Methods: To circumvent insolubility, instability and toxicity of CPT, we used biocompatible, biodegradable and targeted sterically stabilized micelles (SSM) as nanocarriers for CPT (CPT-SSM). We also surface-modified CPT-SSM with vasoactive intestinal peptide (VIP) for active targeting. We then determined whether this nanomedicine abrogated collagen-induced arthritis (CIA) in mice.

Results: Based on our findings, this is the first study to report that CPT was found to be efficacious against CIA at concentrations significantly lower than usual anti-cancer dose. Furthermore, a single subcutaneous injection of CPT-SSM-VIP (0.1 mg/kg) administered to CIA mice mitigated joint inflammation for at least 32 days thereafter without systemic toxicity. CPT alone needed at least 10-fold higher dose to achieve the same effect, albeit with some vacuolization in liver histology.

Conclusion: We propose that CPT-SSM-VIP is a promising targeted nanomedicine and should be further developed as a safe, long-acting, disease-modifying pharmaceutical product for RA.

Fig. 1

CPT was effective against CIA, and efficacy was increased when CPT was delivered as CPT-SSM. Change in (a) paw thickness and (b) clinical arthritis scores of CIA mice with time. Injected with (●) 1 mg/kg CPT alone, (○) 0.3 mg/kg CPT-SSM, (▼) solvent-buffer, and (△) SSM. Paw thickness measurements and clinical arthritis scores were expressed as change from Day 28 values (day treatment injected). *p<0.05 significant reduction versus Day 28 (day treatment injected). Cumulative area under the curves of (c) paw thickness versus time and (d) clinical arthritis score versus time of CIA mice treated with (▮) CPT-SSM or ( ) CPT alone. Cumulative areas under the curve were calculated to indicate the relative extents of CIA symptoms. † p< 0.05 CPT-SSM versus SSM; ‡ p<0.05 CPT versus solvent-buffer; § p<0.05 versus normal mice; ¶ p<0.05 CPT-SSM versus CPTalone. N.S. indicates not significantly different (p>0.05). Results are expressed as mean ± S.E.M. (6 mice/group).

Fig. 2

CPT-SSM-VIP exerts anti-CIA action at lower CPT doses. CPT dose in all drug groups was 0.1 mg/kg. (a) CIA mice paws at the end of the study relative to normal unimmunized DBA/1 mice paw thickness. (b) Clinical arthritis scores at the end of the study relative to normal unimmunized DBA/1 mice at the end of the study. Results are expressed as mean ± S.E. M. (6 mice/group). * p<0.05 CPT-SSM versus CPT-SSM-VIP, † p<0.05 CPT versus CPT-SSM-VIP, ‡ p<0.05 versus normal mice.

Fig. 3

CPT-SSM-VIP abrogates CIA-characteristic inflammation of the synovial tissue, cartilage destruction and bone erosion. (a) Representative histopathology joint section of CPT-SSM-VIP-treated (left) and SSM-VIP control (right) mice on Day 60 (magnification ×100). Arrows represent abnormal infiltration of inflammatory cells and bone/cartilage destruction. (b) Average pooled histological scores of paw sections taken from mice injected with various treatments at effective dose levels and controls. (c) Representative radiographs of CPT-SSM-VIP-treated (left) and SSM-VIP control (right) mice on Day 60. (d) Average pooled radiological scores of paw sections taken from mice injected with various treatments at effective dose levels and controls. Results are expressed as mean ± S.E.M. (6 mice/group). *p<0.05 versus normal mice, †p<0.05 versus empty controls.

Fig. 4

Single-dose MTX and irinotecan did not result in significant reduction in CIA. Change in (A) paw thickness and (B) clinical arthritis score of CIA mice with time. Injected with (●) 1 mg/kg MTX, (○) 10 mg/kg MTX, (▼) irinotecan (0.3 mg/kg) and (△) solvent-buffer. Paw thickness measurements and clinical arthritis scores were expressed as change from Day 28 values (day treatment injected). Results are expressed as mean ± S.E.M. (6 mice/group).

Fig. 5

CPT in micelles reduces cellular content within the CIA joints. Representative joint sections of (A) empty SSM-VIP-injected mice, (B) CPT-SSM-VIP-treated mice; (left to right) CD3+, CD3−, lysozyme+, lysozyme− staining. Bars represent 10 μm. (C) Average pooled immunohistochemical scores of cellular infiltration and distribution in joints of CIA mice on ( ) Day 38 and (▮) Day 60. Results are expressed as mean ± S.E.M. (6 mice/group). *, † p<0.05 versus SSM-VIP and SSM, respectively.

Fig. 6

Representative H&E sections of major organs. (A) spleen, (B) lung, (C) kidney, (D) bone marrow, (E) liver from mice injected with CPT-SSM; (F) liver from mice injected with free CPT. Bar represents 50 μm.